Candida albicans gene, integrin-like protein, antibodies, and methods of use
An isolated and purified DNA molecule encoding Candida albicans protein with integrin-like motifs, the protein itself, antibodies thereto, and methods of use, are provided.
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This application is a divisional of pending U.S. patent application Ser. No. 09/264,604, filed Mar. 8, 1999, now U.S. Pat. No. 6,346,411, which is a divisional of U.S. patent application Ser. No. 08/642,846, filed May 3, 1996, and issued Mar. 23, 1999 as U.S. Pat. No. 5,886,151, both of which are incorporated herein by reference.STATEMENT OF GOVERNMENT RIGHTS
This invention was made with government support under Grant No. R-01 AI25827, awarded by the National Institutes of Health. The government has certain rights in the present invention.BACKGROUND OF THE INVENTION
Candida albicans is the leading fungal pathogen in normal hosts and in patients with damaged immune systems. In normal hosts, disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis). It is estimated that 5% of women of child-bearing age will suffer from recurrent candidal vaginitis (Hurley, “Trends in candidal vaginitis.” Proc. R. Soc. Med. 70 (Suppl. 4), 1-8 (1970), and that virtually every woman will experience at least one episode during her reproductive years. Vaginitis is particularly frequent in otherwise normal females with diabetes or a history of prolonged antibiotic or oral contraceptive use. While short-term topical therapy is effective in treating individual episodes of vaginitis, such agents do not prevent recurrences. Thus, even in the normal host, infection with C. albicans can occur at epithelial surfaces, and recurrences are not prevented by presently available therapies.
In immunocompromised hosts such as cancer patients, transplant patients, post-operative surgical patients, premature newborns, or HIV-infected people, C. albicans ranks as the leading fungal pathogen. In this population, disease ranges from aggressive local infections such as periodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone. Such grave prognoses require more toxic therapy, with attendant consequences from both the underlying infection and the treatment. Here again, the infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression. Strategies to interrupt candidal adhesion therefore have broad applicability to the prevention of mild but recurrent disease in the normal host and to the reduction of substantial morbidity and mortality in the immunocompromised.
It is well recognized that C. albicans adheres to epithelial and endothelial cells in the human host, oftentimes by recognizing proteins of the extracellular matrix called ligands. These ligands include proteins such as fibronectin, vitronectin, fibrinogen, the C3 degradation fragment iC3b, or the shorter C3 degradation fragment C3d. Because recognition of all of these proteins except C3d is dependent upon the amino acid sequence ARGININE-GLYCINE-ASPARTIC ACID or R-G-D, these candidal adhesions are thought to operate like the vertebrate integrins and are called “integrin-like proteins” or “integrin analogs.”
Vertebrate integrins are composed of two subunits: an &agr;-subunit and a &bgr;-subunit. There are approximately 14 &agr; and 8 &bgr;subunits described to date in vertebrate cells. Using monoclonal or polyclonal antibodies to vertebrate integrins, several investigators have obtained evidence for integrin-like proteins in C. albicans: an &agr;M analog, an &agr;5/&bgr;1 complex, or a &bgr;1 analog. Neither the &agr;5/&bgr;1 complex nor the &bgr;1 analog has been isolated from C. albicans or from any other candidal species, and the responsible genes encoding these “integrin-like proteins” have not been identified.SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule encoding a Candida albicans protein with integrin-like motifs that hybridizes to DNA complementary to DNA having SEQ ID NO:1 under the stringency conditions of hybridization in buffer containing 5×SSC, 5×Denhardt's, 0.5% SDS, 1 mg salmon sperm/25 mls of hybridization solution incubated at 65° C. overnight, followed by high stringency washing with 0.2×SSC/0.1 % SDS at 65° C. Preferably, the present invention provides an isolated and purified DNA molecule encoding the Candida albicans protein with integrin-like motifs which has the amino acid sequence having SEQ ID NO:2. Preferably, the DNA is genomic DNA which has the nucleotide sequence shown in Table 1 (SEQ ID NO:1).
The present invention also provides a vector and a cell line transformed by an extrachromosomal plasmid containing non-native DNA encoding Candida albicans protein with integrin-like motifs (i.e., C. albicans integrin-like protein), as described herein. The cell line preferably comprises S. cerevisiae. This cell line can be used in a method of delivering a gene product to a subject.
The present invention also provides a Candida albicans protein with integrin-like motifs comprising an I domain, two EF-hand divalent cation binding sites, a sequence sufficient to encode a transmembrane domain, an internal RGD tripeptide, and a carboxy-terminal sequence with a single tyrosine residue. As used herein, an “internal” RGD tripeptide means that the RGD sequence is in the Candida protein, not in the vertebrate proteins recognized by integrins. Preferably, the isolated and purified C. albicans integrin-like protein has an amino acid sequence which is SEQ ID NO:2. Also provided are isolated and purified peptides, such as those having an amino acid sequence selected from the group consisting of:YLS PTN NNN SKN VSD MDL HLQ NL; (SEQ ID NO:4) DWK LED SND GDR EDN DDI SRF EK; (SEQ ID NO:5) SKS ANT VRG DDD GLA SA; (SEQ ID NO:6) DHL DSF DRS YNH TEQ SI; and (SEQ ID NO:7) WIQ NLQ EII YRN RFR RQ. (SEQ ID NO:8)
The invention also provides a vaccine comprising the protein and peptides, either singly or together, described herein as well as an isolated and purified antibodies to the C. albicans integrin-like protein and peptides described herein.
The invention also provides a method of inhibiting adhesion of Candida albicans to cells (preferably epithelial cells, and more preferably human epithelial cells). The method includes contacting the Candida albicans with antibodies to the Candida albicans protein with integrin-like motifs (&agr;Int1p) or to fragments thereof as described herein.BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the blockade of candidal adhesion to HeLa cells by antibodies to &agr;Int1p.
FIG. 2 is a graph of the blockade of candidal adhesion to CHO cells by antibodies to &agr;Intp1.DETAILED DESCRIPTION
Specifically, the present invention is directed to the cloning and expression of a gene (&agr;INT1) for an integrin-like protein (&agr;Int1p) of Candida albicans. To that end, the invention provides an isolated and purified DNA molecule encoding a Candida albicans protein with an integrin-like motifs or biologically active derivative thereof. More preferably, the DNA is a genomic DNA molecule that encodes the protein represented by the amino acid sequence shown in Table 2 (SEQ ID NO:2). Most preferably, the genomic DNA molecule is represented by the complete nucleotide sequence shown in Table 1 (SEQ ID NO:1). Isolated and purified peptides encoded by this DNA, and derivatives thereof, which are biologically active are also within the scope of the invention.
As used herein, the terms “isolated and purified” refer to in vitro isolation of a DNA molecule or protein from its natural cellular environment, and from association with other coding regions of the C. albicans genome, so that it can be sequenced, replicated, and/or expressed. Preferably, the isolated and purified DNA molecules of the invention comprise a single coding region. Thus, the present DNA molecules are those consisting essentially of a DNA segment encoding an integrin-like protein or biologically active derivative thereof. Although the DNA molecule includes a single coding region, it can contain additional nucleotides that do not detrimentally affect the function of the DNA molecule, i.e., the expression of the integrin-like protein or biologically active derivative thereof. For example, the 5′ and 3′ untranslated regions may contain variable numbers of nucleotides. Preferably, additional nucleotides are outside the single coding region.
The present invention also provides an isolated and purified DNA molecule that encodes integrin-like protein (&agr;Int1p) and that hybridizes to a DNA molecule complementary to the DNA molecule shown in Table 1 (SEQ ID NO:1) under high stringency hybridization conditions. As used herein, “high stringency hybridization conditions” refers to hybridization in buffer containing 5×SSC, 5×Denhardt's, 0.5% SDS, 1 mg salmon sperm/25 mls of hybridization solution incubated at 65° C. overnight, followed by high stringency washing with 0.2×SSC/0.1% SDS at 65° C.
It is envisioned that oligonucleotides are also possible. Oligonucleotide probes and primers are segments of labeled, single-stranded DNA which will hybridize, or noncovalently bind, with complementary single-stranded DNA to be identified.
If desired, the probe and primer can be labeled with any suitable label known to those skilled in the art, including radioactive and nonradioactive labels. Typical radioactive labels include 32P, 125I, 35S, and the like. Nonradioactive labels include, for example, ligands such as biotin or digoxigenin as well as enzymes such as phosphatase or peroxidases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probe or primer may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at one end and a biotin label at the other end.
As used herein, the terms “protein with integrin-like motifs” and “integrin-like protein” refer to a candidal adhesin of C. albicans, that is expressed at the surface of C. albicans and allows candida to bind to epithelial cells, for example. This initial adhesion to epithelium leads to subsequent events in the pathogenesis of invasive candidal infection (e.g., penetration of epithelial barriers and hematogenous dissemination). The unmodified protein (i.e., prior to any post-translational modification) is preferably of about 180-190 kDa, and more preferably of about 188 kDa. It includes several motifs common to &agr;M and &agr;X leukocyte integrins. These motifs include: (1) an Inserted domain (“I” domain) containing a conformationally dependent cation binding site (or MIDAS motif, as disclosed in Michishita et al., Cell, 72, 857-867 (1993)); (2) two linear divalent cation binding sites conforming to the EF-hand motif; (3) a sequence sufficient to encode a transmembrane domain; (4) a carboxy-terminal sequence with a single tyrosine residue; and (5) an internal RGD tripeptide (arginine-glycine-aspartic acid). The RGD site is at amino acids 1149-1151 in SEQ ID NO:2.
A “biologically active derivative thereof” is an integrin-like protein that is modified by amino acid deletion, addition, substitution, or truncation, or that has been chemically derivatized, but that nonetheless functions in the same manner as the protein of SEQ ID NO:2. For example, it is known in the art that substitutions of aliphatic amino acids such as alanine, valine and isoleucine with other aliphatic amino acids can often be made without altering the structure or function of a protein. Similarly, substitution of aspartic acid for glutamic acid, in regions other than the active site of an enzyme, are likely to have no appreciable affect on protein structure or function. The term “biologically active derivative” is intended to include C. albicans proteins with integrin-like motifs as thus modified. The term also includes fragments, variants, analogs or chemical derivatives thereof. The term “fragment” is meant to refer to any polypeptide subset. Fragments can be prepared by subjecting C. albicans proteins with integrin-like motifs to the action of any one of a number of commonly available proteases, such as trypsin, chymotrypsin or pepsin, or to chemical cleavage agents, such as cyanogen bromide. The term “variant” is meant to refer to a molecule substantially similar in structure and function to either the entire C. albicans integrin-like protein or to a fragment thereof. A protein or peptide is said to be “substantially similar” if both molecules have substantially similar amino acid sequences, preferably greater than about 80% sequence identity, or if the three-dimensional backbone structures of the molecules are superimposable, regardless of the level of identity between the amino acid sequences. Thus, provided that two molecules possess similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical. The term “analog” is meant to refer to a protein that differs structurally from the wild type C. albicans integrin-like protein, but possesses similar activity.
Several fragments of the protein have been prepared and can be used in vaccines or as antigens to prepare anti-peptide antibodies, which can be monoclonal or polyclonal (preferably polyclonal). A 236 amino acid sequence near the amino terminus of the gene product (&agr;Int1p) is shown in Table 3 (SEQ ID NO:3). A 23-mer peptide encompassing the first cation-binding site is YLS PTN NNN SKN VSD MDL HLQ NL (SEQ ID NO:4). A 23-mer peptide encompassing the second divalent cation-binding site is DWK LED SND GDR EDN DDI SRF EK (SEQ ID NO:5). A 17-mer peptide spanning the RGD site and flanking residues is SKS ANT VRG DDD GLA SA (SEQ ID NO:6). A 17-mer peptide from the MIDAS motif of &agr;Int1p is DHL DSF DRS YNH TEQ SI (SEQ ID NO:7). A 17-mer peptide from the C-terminus of &agr;Int1p is WIQ NLQ EII YRN RFR RQ (SEQ ID NO:8).
The antibodies produced to these peptides bind to C. albicans blastospores, germ tubes, and hyphae, and thereby block epithelial adhesion of C. albicans (i.e., candida). Preferably, the antibodies are able to block C. albicans adhesion by at least about 30%, and preferably by at least about 50%. It is believed that this blocking activity of the initial adhesion to epithelium will reduce and even prevent subsequent events in the pathogenesis of invasive candidal infection.
The present invention also provides a vector comprising an isolated and purified DNA molecule encoding C. albicans protein with integrin-like motifs or a biologically active derivative thereof, preferably C. albicans protein with integrin-like motifs having the amino acid sequence of SEQ ID NO:2. Preferably, the vector includes a sequence encoding the C. albicans protein with integrin-like motifs as well as a second DNA segment operably linked to the coding sequence and capable of directing expression of the coding region, such as a promoter region operably linked to the 5′ end of the coding DNA sequence. The vector can also include a DNA segment that is a selectable marker gene or a reporter gene as well as upstream untranslated sequence from the C. albicans gene.
The present invention also provides a cell line, preferably a Saccharomyces cerevisiae yeast strain transformed with an extrachromosomal plasmid containing non-native DNA encoding the C. albicans protein with integrin-like motifs. S. cerevisiae, also known as brewer's yeast or baker's yeast, typically exhibits a spheriod, yeast-like form and, under certain conditions, can also exhibit a filamentous, mold-like form. The filamentous cells, which are often referred to as pseudohyphal cells, have an elongated morphology. S. cerevisiae (preferably haploid S. cerevisiae), which is seldom a pathogen, transformed with the open reading frame of &agr;Int1, displays germ tube-like projections referred to herein as “noses.” Thus, synthesis of the Candida gene product &agr;Int1p in S. cerevisiae induces germ tubes. Furthermore, &agr;Int1p is surface expressed in S. cerevisiae and can be recognized by polyclonal antibodies to &agr;Int1p peptides and by monoclonal antibodies to vertebrate integrins. In this way, a generally harmless yeast becomes “sticky” and “nosey.”
The S. cerevisiae yeast cells transformed by the gene described herein will adhere to epithelial surfaces as a result of expression of the integrin-like gene described herein; however, they will not invade the cells. Thus, “sticky” S. cerevisiae may colonize in patients at risk for Candida infection and thereby block the adhesion sites, and reduce or eliminate the opportunity for Candida to adhere, colonize, and invade. Also, the “sticky” S. cerevisiae may function as a gene or gene product delivery system. For example, it is envisioned that a phosphate-binding protein could be delivered to the gastrointestinal tract of a patient with chronic renal failure using Saccharomyces transformed by the integrin-like gene and a second plasmid for expression of the phosphate-binding protein. Alternatively, a second plasmid could be used to provide a source of vaccine antigen for gastrointestinal pathogens like cholera. In the genitourinary tract, expression of spermicides by S. cerevisiae transformed with the C. albicans integrin-like gene on an extrachromosomal plasmid could provide a cheap and infrequent method of contraception. Also, synthesis of protein-based antiretroviral agents could help to reduce transmission of HIV in the birth canal.
1. Isolation of DNA
Several different methods are available for isolating genomic DNA. Most approaches begin with the purification of protein. Purified protein is then subjected to amino acid microsequencing, either directly or after limited cleavage. The partial amino acid sequence that is obtained can be used to design degenerate oligonucleotide probes or primers for use in the generation of unique, nondegenerate nucleotide sequences by polymerase chain reaction (PCR), sequences that can in turn be used as probes for screening genomic DNA libraries. Antibodies raised against purified protein may also be used to isolate DNA clones from expression libraries.
Alternatively, the sequences of DNAs for related proteins (e.g., human integrins) may be used as starting points in a cloning strategy, so-called “cloning by homology”. Another way of utilizing sequence information from different species is to take advantage of shorter areas of high sequence homology among related DNAs from different species and to perform PCR to obtain “species-specific” nondegenerate nucleotide sequences. Such a sequence can then be used for library screening or even for direct PCR-based cloning. Detection of the desired DNA can also involve the use of PCR using novel primers.
Alternatively, the region encoding &agr;Int1p may be obtained from a genomic DNA library or by in vitro polynucleotide synthesis from the complete nucleotide acid sequence.
Libraries are screened with appropriate probes designed to identify the genomic DNA of interest. Preferably, for genomic libraries, suitable probes include oligonucleotides that consist of known or suspected portions of the &agr;Int1p genomic DNA from the same or different species; and/or complementary or homologous genomic DNAs or fragments thereof that consist of the same or a similar DNA. For expression libraries (which express the protein), suitable probes include monoclonal or polyclonal antibodies that recognize and specifically bind to the &agr;Int1p protein. Appropriate probes for screening genomic DNA libraries include, but are not limited to, oligonucleotides, genomic DNAs, or fragments thereof that consist of the same or a similar gene, and/or homologous genomic DNAs or fragments thereof. Screening the genomic DNA library with the selected probe may be accomplished using standard procedures.
Screening genomic DNA libraries using synthetic oligonucleotides as probes is a preferred method of practicing this invention. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous to minimize false positives. The actual nucleotide sequence(s) of the probe(s) is usually designed based on regions of the &agr;Int1p genomic DNA that have the least codon redundancy. The oligonucleotides may be degenerate at one or more positions, i.e., two or more different nucleotides may be incorporated into an oligonucleotide at a given position, resulting in multiple synthetic oligonucleotides. The use of degenerate oligonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known.
The oligonucleotide can be labeled such that it can be detected upon hybridization to DNA in the library being screened. A preferred method of labeling is to use ATP and polynucleotide kinase to radiolabel the 5′ end of the oligonucleotide. However, other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling.
Of particular interest is the &agr;Int1 nucleotide sequence that encodes a full-length mRNA transcript, including the complete coding region for the gene product, &agr;Int1p. Nucleic acid containing the complete coding region can be obtained by screening selected genomic DNA libraries using an oligonucleotide encoding the deduced amino acid sequence.
An alternative means to isolate the DNA encoding &agr;Int1p is to use PCR methodology. This method requires the use of oligonucleotide primer probes that will hybridize to the DNA encoding &agr;Int1p. Strategies for selection of PCR primer oligonucleotides are described below.
2. Insertion of DNA into Vector
The nucleic acid containing the &agr;Int1coding region is preferably inserted into a replicable vector for further cloning (amplification of the DNA) or for expression of the gene product. Many vectors are available, and selection of the appropriate vector will depend on: 1) whether it is to be used for DNA amplification or for DNA expression; 2) the size of the nucleic acid to be inserted into the vector; and 3) the host cell to be transformed with the vector. Most expression vectors are “shuttle” vectors, i.e., they are capable of replication in at least one class of organism but can be transfected into another organism for expression. For example, a vector replicates in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome. Each replicable vector contains various structural components depending on its function (amplification of DNA or expression of DNA) and the host cell with which it is compatible. These components are described in detail below.
Construction of suitable vectors employs standard ligation techniques known in the art. Isolated plasmid or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. Typically, the ligation mixtures are used to transform E. coli K12 or E. coli XL1 Blue MRF strains 294 (ATCC 31,446) and successful transformants are selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by methods known in the art.
Replicable cloning and expression vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.
Vector component: origin of replication. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.
Vector component: marker gene. Expression and cloning vectors may contain a marker gene, also termed a selection gene or selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, streptomycin or tetracycline; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacillus. One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive the selection regimen.
A suitable marker gene for use in yeast is URA3 or the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282, 39 (1979); Kingsman et al., Gene, 7, 141 (1979); or Tschemper et al., Gene, 10, 157 (1980)). The TRP1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85, 23 (1977)).
Vector component: promoter. Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the gene. Promoters are untranslated sequences located upstream (5′) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of a particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. In contrast, constitutive promoters produce a constant level of transcription of the cloned DNA segment.
At this time, a large number of promoters recognized by a variety of potential host cells are well known in the art. Promoters are removed from their source DNA using a restriction enzyme digestion and inserted into the cloning vector using standard molecular biology techniques. Native or heterologous promoters can be used to direct amplification and/or expression of DNA. Heterologous promoters are preferred, as they generally permit greater transcription and higher yields of expressed protein as compared to the native promoter. Well-known promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Such promoters can be ligated to the DNA to be expressed using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems may contain a Shine-Dalgarno sequence for RNA polymerase binding.
Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bp upstream from the site where transcription is initiated Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CXCAAT region where X may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be a signal for addition of the poly A tail to the 3′ end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
Vector component: enhancer element. Transcription of DNA by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually having about 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation-and position-independent, having been found 5′ and 3′ to the transcription unit, within an intron as well as within the coding sequence itself. Typically, an enhancer from a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to the DNA, but is preferably located at a site 5′ of the promoter.
Vector component: transcription termination. Expression vectors used in eukaryotic host cells (e.g., yeast, fungi, etc.) can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally, 3′ untranslated regions of eukaryotic or viral DNAs. These regions can contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA.
The genetically engineered plasmid of the invention can be used to transform a host cell. As discussed above, a particularly desirable host is a eukaryotic microbe such as filamentous fungi or yeast. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis, K. bulgaricus, K. thermotolerans, and K. marxianus, yarrowia, Pichia pastoris, Trichoderma reesia, Neurospora crassa, and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans.
4. Transfection and Transformation
Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequence are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, the calcium phosphate precipitation method and electroporation are commonly used. Successful transfection is generally recognized when any indication of the operation of the vector occurs within the host cell.
Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130, 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76(8) 3829-3833 (1979). However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used.
5. Cell Culture
Cells used to produce the &agr;Int1gene product are cultured in suitable media, as described generally in Sambrook et al. Commercially available media such as Hams F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. These media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin' drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose, galactose, or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. Induction of cells, to cause expression of the protein, is accomplished using the procedures required by the particular expression system selected.
The invention has been described with reference to various specific and preferred embodiments and will be further described by reference to the following detailed examples. It is understood, however, that there are many extensions, variations and modifications on the basic theme of the present invention beyond that shown in the examples and detailed description, which are within the spirit and scope of the present invention.EXAMPLES
Isolation of the Gene &agr;Int1 from Candida albicans
DNA from spheroplasts of C. albicans 10261 (American Type Culture Collection) was isolated according to standard procedures as disclosed in Davis et al., Methods Enzymol., 65, 404-411 (1980), digested with the restriction enzyme Sau3AI, and packaged in &lgr;EMBL3 (Stratagene). Preliminary studies confirmed that a 3.5 kbp EcoRI fragment of C. albicans DNA hybridized with a 314 bp EcoRI/SmaI DNA fragment derived from the transmembrane domain of human &agr;M as disclosed in Hickstein et al., Proc. Natl. Acad. Sci. USA, 86, 257-261, 1989. Primers for amplification of the EcoRI/SmaI &agr;M DNA fragment were as follows:upstream primer: 5′ GAATTCAATGCTACCCTCAA; (SEQ ID NO:9) downstream primer: 5′ CCCGGGGGACCCCCTTCACT. (SEQ ID NO:10)
A library enriched for 3.0-3.8 kbp EcoRI fragments from C. albicans was constructed by digestion of genomic DNA with EcoRI and ligation to pBluescript II SK(+). Plasmid minipreparations from a total of 200 colonies were screened by the sib selection technique for hybridization at 50° C. with [32P]-labeled PCR product. Five clones were isolated from three successive screenings. Two of the five clones gave reproducible signals after hybridization with a degenerate oligonucleotide encoding a conserved sequence [KVGFFK] in the cytoplasmic domain of &agr;X: 5′ AA(AG) GT(CT) GG(AT) TT(CT) TT(CT) AA(AG) 3′ (SEQ ID NO:11). Both clones contained a 3.5 kbp EcoRI insert and failed to hybridize with a degenerate oligonucleotide from the S. cerevisiae gene USO1:5′ GAA AT(ACT) GA(CT) GA(CT) TT(AG) ATG 3′ (SEQ ID NO:12).
A 500 bp HindIII subfragment from one of these clones was used to screen 20,000 clones from a library of C. albicans 10261 genomic DNA (prepared commercially from C. albicans DNA by Stratagene) by the plaque hybridization technique as disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y., 2nd Ed., pp.2.108-2.125 (1989). The largest hybridizing insert, a 10.5 kbp SalI fragment, was isolated by agarose gel electrophoresis, cloned, and sequenced.
Both strands of the 10.5 kbp SalI fragment were sequenced by the method of gene walking on an Applied Biosystems Model 373 Automated Sequencer in the University of Minnesota Microchemical Facility. Nucleotide and protein sequence analyses were performed with the Genetics Computer Group (U. of WI, Madison) Sequence Analysis Software Package, version 7.0. The nucleotide sequence of the coding strand plus approximately 100 upstream nucleotides and 100 nucleotides of 3′ untranslated sequence and the derived amino acid sequence (GenBank Acc. No. U35070) are shown in Tables 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2), respectively. By Southern blot analysis under conditions of high stringency (hybridization at 65° C., final wash in 0.2×SSC/0.1% SDS at 65° C.), this gene is present only in C. albicans and not in strains of C. tropicalis, C. krusei, C. glabrata, or S. cerevisiae.TABLE 1 1 cccaaaaaag ataaaataaa aacaaaacaa aacaaaagta ctaacaaatt attgaaactt 61 ttaattttta ataaagaatc agtagatcta ttgttaaaag aaatgaactc aactccaagt 121 aaattattac cgatagataa acattctcat ttacaattac agcctcaatc gtcctcggca 181 tcaatattta attccccaac aaaaccattg aatttcccca gaacaaattc caagccgagt 241 ttagatccaa attcaagctc tgatacctac actagcgaac aagatcaaga gaaagggaaa 301 gaagagaaaa aggacacagc ctttcaaaca tcttttgata gaaattttga tcttgataat 361 tcaatcgata tacaacaaac aattcaacat cagcaacaac agccacaaca acaacaacaa 421 ctctcacaaa ccgacaataa tttaattgat gaattttctt ttcaaacacc gatgacttcg 481 actttagacc taaccaagca aaatccaact gtggacaaag tgaatgaaaa tcatgcacca 541 acttatataa atacctcccc caacaaatca ataatgaaaa aggcaactcc taaagcgtca 601 cctaaaaaag ttgcatttac tgtaactaat cccgaaattc atcattatcc agataataga 661 gtcgaggaag aagatcaaag tcaacaaaaa gaagattcag ttgagccacc cttaatacaa 721 catcaatgga aagatccttc tcaattcaat tattctgatg aagatacaaa tgcttcagtt 781 ccaccaacac caccacttca tacgacgaaa cctacttttg cgcaattatt gaacaaaaac 841 aacgaagtca atctggaacc agaggcattg acagatatga aattaaagcg cgaaaatttc 901 agcaatttat cattagatga aaaagtcaat ttatatctta gtcccactaa taataacaat 961 agtaagaatg tgtcagatat ggatctgcat ttacaaaact tgcaagacgc ttcgaaaaac 1021 aaaactaatg aaaatattca caatttgtca tttgctttaa aagcaccaaa gaatgatatt 1081 gaaaacccat taaactcatt gactaacgca gatattctgt taagatcatc tggatcatca 1141 caatcgtcat tacaatcttt gaggaatgac aatcgtgtct tggaatcagt gcctgggtca 1201 cctaagaagg ttaatcctgg attgtctttg aatgacggca taaaggggtt ctctgatgag 1261 gttgttgaat cattacttcc tcgtgactta tctcgagaca aattagagac tacaaaagaa 1321 catgatgcac cagaacacaa caatgagaat tttattgatg ctaaatcgac taataccaat 1381 aagggacaac tcttagtatc atctgatgat catttggact cttttgatag atcctataac 1441 cacactgaac aatcaatttt gaatcttttg aatagtgcat cacaatctca aatttcgtta 1501 aatgcattgg aaaaacaaag gcaaacacag gaacaagaac aaacacaagc ggcagagcct 1561 gaagaagaaa cttcgtttag tgataatatc aaagttaaac aagagccaaa gagcaatttg 1621 gagtttgtca aggttaccat caagaaagaa ccagttctgg ccacggaaat aaaagctcca 1681 aaaagagaat tttcaagtcg aatattaaga ataaaaaatg aagatgaaat tgccgaacca 1741 gctgatattc atcctaaaaa agaaaatgaa gcaaacagtc atgtcgaaga tactgatgca 1801 ttgttgaaga aagcacttaa tgatgatgag gaatctgaca cgacccaaaa ctcaacgaaa 1861 atgtcaattc gttttcatat tgatagtgat tggaaattgg aagacagtaa tgatggcgat 1921 agagaagata atgatgatat ttctcgtttt gagaaatcag atattttgaa cgacgtatca 1981 cagacttctg atattattgg tgacaaatat ggaaactcat caagtgaaat aaccaccaaa 2041 acattagcac ccccaagatc ggacaacaat gacaaggaga attctaaatc tttggaagat 2101 ccagctaata atgaatcatt gcaacaacaa ttggaggtac cgcatacaaa agaagatgat 2161 agcattttag ccaactcgtc caatattgct ccacctgaag aattgacttt gcccgtagtg 2221 gaagcaaatg attattcatc ttttaatgac gtgaccaaaa cttttgatgc atactcaagc 2281 tttgaagagt cattatctag agagcacgaa actgattcaa aaccaattaa tttcatatca 2341 atttggcata aacaagaaaa gcagaagaaa catcaaattc ataaagttcc aactaaacag 2401 atcattgcta gttatcaaca atacaaaaac gaacaagaat ctcgtgttac tagtgataaa 2461 gtgaaaatcc caaatgccat acaattcaag aaattcaaag aggtaaatgt catgtcaaga 2521 agagttgtta gtccagacat ggatgatttg aatgtatctc aatttttacc agaattatct 2581 gaagactctg gatttaaaga tttgaatttt gccaactact ccaataacac caacagacca 2641 agaagtttta ctccattgag cactaaaaat gtcttgtcga atattgataa cgatcctaat 2701 gttgttgaac ctcctgaacc gaaatcatat gctgaaatta gaaatgctag acggttatca 2761 gctaataagg cagcgccaaa tcaggcacca ccattgccac cacaacgaca accatcttca 2821 actcgttcca attcaaataa acgagtgtcc agatttagag tgcccacatt tgaaattaga 2881 agaacttctt cagcattagc accttgtgac atgtataatg atatttttga tgatttcggt 2941 gcgggttcta aaccaactat aaaggcagaa ggaatgaaaa cattgccaag tatggataaa 3001 gatgatgtca agaggatttt gaatgcaaag aaaggtgtga ctcaagatga atatataaat 3061 gccaaacttg ttgatcaaaa acctaaaaag aattcaattg tcaccgatcc cgaagaccga 3121 tatgaagaat tacaacaaac tgcctctata cacaatgcca ccattgattc aagtatttat 3181 ggccgaccag actccatttc taccgacatg ttgccttatc ttagtgatga attgaaaaaa 3241 ccacctacgg ctttattatc tgctgatcgt ttgtttatgg aacaagaagt acatccgtta 3301 agatcaaact ctgttttggt tcacccaggg gcaggagcag caactaattc ttcaatgtta 3361 ccagagccag attttgaatt aatcaattca cctgctagaa atgtgctgaa caacagtgat 3421 aatgtcgcca tcagtggtaa tgctagtact attagtttta accaattgga tatgaatttt 3481 gatgaccaag ctacaattgg tcaaaaaatc caagagcaac ctgcttcaaa atccgccaat 3541 actgttcgtg gtgatgatga tggattggcc agtgcacctg aaacaccaag aactcctacc 3601 aaaaaggagt ccatatcaag caagcctgcc aagctttctt ctgcctcccc tagaaaatca 3661 ccaattaaga ttggttcacc agttcgagtt attaagaaaa atggatcaat tgctggcatt 3721 gaaccaatcc caaaagccac tcacaaaccg aagaaatcat tccaaggaaa cgagatttca 3781 aaccataaag tacgagatgg tggaatttca ccaagctccg gatcagagca tcaacagcat 3841 aatcctagta tggtttctgt tccttcacag tatactgatg ctacttcaac ggttccagat 3901 gaaaacaaag atgttcaaca caagcctcgt gaaaagcaaa agcaaaagca tcaccatcgc 3961 catcatcatc atcatcataa acaaaaaact gatattccgg gtgttgttga tgatgaaatt 4021 cctgatgtag gattacaaga acgaggcaaa ttattcttta gagttttagg aattaagaat 4081 atcaatttac ccgatattaa tactcacaaa ggaagattca ctttaacgtt ggataatgga 4141 gtgcattgtg ttactacacc agaatacaac atggacgacc ataatgttgc cataggtaaa 4201 gaatttgagt tgacagttgc tgattcatta gagtttattt taactttgaa ggcatcatat 4261 gaaaaacctc gtggtacatt agtagaagtg actgaaaaga aagttgtcaa atcaagaaat 4321 agattgagtc gattatttgg atcgaaagat attatcacca cgacaaagtt tgtgcccact 4381 gaagtcaaag atacctgggc taataagttt gctcctgatg gttcatttgc tagatgttac 4441 attgatttac aacaatttga agaccaaatc accggtaaag catcacagtt tgatctcaat 4501 tgttttaatg aatgggaaac tatgagtaat ggcaatcaac caatgaaaag aggcaaacct 4561 tataagattg ctcaattgga agttaaaatg ttgtatgttc cacgatcaga tccaagagaa 4621 atattaccaa ccagcattag atccgcatat gaaagcatca atgaattaaa caatgaacag 4681 aataattact ttgaaggtta tttacatcaa gaaggaggtg attgtccaat ttttaagaaa 4741 cgttttttca aattaatggg cacttcttta ttggctcata gtgaaatatc tcataaaact 4801 agagccaaaa ttaatttatc aaaagttgtt gatttgattt atgttgataa agaaaacatt 4861 gatcgttcca atcatcgaaa tttcagtgat gtgttattgt tggatcatgc attcaaaatc 4921 aaatttgcta atggtgagtt gattgatttt tgtgctccta ataaacatga aatgaaaata 4981 tggattcaaa atttacaaga aattatctat agaaatcggt tcagacgtca accatgggta 5041 aatttgatgc ttcaacaaca acaacaacaa caacaacaac aaagctccca acagtaattg 5101 aaaggtctac ttttgatttt tttaatttta attggcaaat atatgcccat tttgtattat 5161 cttttagtct aatagcgttt tctttttttc cagt TABLE 2 1 MNSTPSKLLPIDKHSHLQLQPQSSSASIFNSPTKPLNFPRTNSKPSLDPN 51 SSSDTYTSEQDQEKGKEEKKDTAFQTSFDRNFDLDNSIDIQQTIQHQQQQ 101 PQQQQQLSQTDNNLIDEFSFQTPMTSTLDLTKQNPTVDKVNENHAPTYIN 151 TSPNKSIMKKATPKASPKKVAFTVTNPEIHHYPDNRVEEEDQSQQKEDSV 201 EPPLIQHQWKDPSQFNYSDEDTNASVPPTPPLHTTKPTFAQLLNKNNEVN 251 SEPEALTDMKLKRENFSNLSLDEKVNLYLSPTNNNNSKNVSDMDSHLQNL 301 QDASKNKTNENIHNLSFALKAPKNDIENPLNSLTNADISLRSSGSSQSSL 351 QSLRNDNRVLESVPGSPKKVNPGLSLNDGIKGFSDEVVESLLPRDLSRDK 401 LETTKEHDAPEHNNENFIDAKSTNTNKGQLLVSSDDHLDSFDRSYNHTEQ 451 SILNLLNSASQSQISLNALEKQRQTQEQEQTQAAEPEEETSFSDNIKVKQ 501 EPKSNLEFVKVTIKKEPVSATEIKAPKREFSSRILRIKNEDEIAEPADIH 551 PKKENEANSHVEDTDALLKKALNDDEESDTTQNSTKMSIRFHIDSDWKLE 601 DSNDGDREDNDDISRFEKSDILNDVSQTSDIIGDKYGNSSSEITTKTLAP 651 PRSDNNDKENSKSLEDPANNESLQQQLEVPHTKEDDSILANSSNIAPPEE 701 LTLPVVEANDYSSFNDVTKTFDAYSSFEESLSREHETDSKPINFISIWHK 751 QEKQKKHQIHKVPTKQIIASYQQYKNEQESRVTSDKVKIPNAIQFKKFKE 801 VNVMSRRVVSPDMDDLNVSQFLPELSEDSGFKDLNFANYSNNTNRPRSFT 851 PLSTKNVLSNIDNDPNVVEPPEPKSYAEIRNARRLSANKAAPNQAPPLPP 901 QRQPSSTRSNSNKRVSRFRVPTFEIRRTSSALAPCDMYNDIFDDFGAGSK 951 PTIKAEGMKTLPSMDKDDVKRILNAKKGVTQDEYINAKLVDQKPKKNSIV 1001 TDPEDRYEELQQTASIHNATIDSSIYGRPDSISTDMLPYLSDELKKPPTA 1051 LLSADRLFMEQEVHPLRSNSVLVHPGAGAATNSSMLPEPDFELINSPARN 1101 VSNNSDNVAISGNASTISFNQLDMNFDDQATTGQKIQEQPASKSANTVRG 1151 DDDGLASAPETPRTPTKKESISSKPAKLSSASPRKSPIKIGSPVRVIKKN 1201 GSIAGIEPIPKATHKPKKSFQGNEISNHKVRDGGISPSSGSEHQQHNPSM 1251 VSVPSQYTDATSTVPDENKDVQHKPREKQKQKHHHRHHHHHHKQKTDIPG 1301 VVDDEIPDVGLQERGKLFFRVLGIKNINLPDINTHKGRFTLTLDNGVHCV 1351 TTPEYNMDDHNVAIGKEFELTVADSLEFILTLKASYEKPRGTLVEVTEKK 1401 VVKSRNRLSRLFGSKDIITTTKFVPTEVKDTWANKFAPDGSFARCYIDLQ 1451 QFEDQITGKASQFDLNCFNEWETMSNGNQPMKRGKPYKIAQLEVKMLYVP 1501 RSDPREILPTSIRSAYESINELNNEQNNYFEGYLHQEGGDCPIFKKRFFK 1551 LMGTSLLAHSEISHKTRAKINLSKVVDLIYVDKENIDRSNIRNFSDVLLL 1601 DHAFKIKFANGELIDFCAPNKHEMKIWIQNLQEIIYRNRFRRQPWVNLML 1651 QQQQQQQQQQSSQQ
A 236 amino acid sequence near the amino terminus of the gene product (&agr;Int1p) is shown in Table 3 (SEQ ID NO:3). This sequence, or a portion thereof, is believed to encompass the ligand binding site, or a portion thereof, and would provide very useful antibodies or could be used as a vaccine antigen itself.TABLE 3 SDEDTNASVPPTPPLHTTKPTFAQLLNKNNEVN 251 SEPEALTDMKLKRENFSNLSLDEKVNLYLSPTNNNNSKNVSDMDSHLQNL 301 QDASKNKTNENIHNLSFALKAPKNDIENPLNSLTNADISLRSSGSSQSSL 351 QSLRNDNRVLESVPGSPKKVNPGLSLNDGIKGFSDEVVESLLPRDLSRDK 401 LETTKEHDAPEHNNENFIDAKSTNTNKGQLLVSSDDHLDSPDRSYNHTEQ 451 SIL
The following peptide sequences were used as antigens for the preparation of anti-peptide polyclonal antibodies in rabbits by commercial contract through Cocalico Biologicals (Reamstown, Pa.). The sequences B-F are listed below and correspond to the protein sequence of &agr;Int1p as reported in GenBank, with the exception of one amino acid substitution in sequence (B), as noted below.
B. A 23-mer peptide encompassing the first cation-binding site. This peptide was synthesized by BioSynthesis Inc. (Lewisville, Tex.). Note that the peptide sequence is MDL, while the GenBank sequence is MDS.
YLS PTN NNN SKN VSD MDL HLQ NL (SEQ ID NO:4)
C. A 23-mer peptide encompassing the second divalent cation-binding site. This peptide was synthesized by BioSynthesis Inc. (Lewisville, Tex.).
DWK LED SND GDR EDN DDI SRF EK (SEQ ID NO:5)
D. A 17-mer peptide spanning the RGD site and flanking residues. This peptide was synthesized by the Microchemical Facility of the University of Minnesota.
SKS ANT VRG DDD GLA SA (SEQ ID NO:6)
E. A 17-mer peptide from the MIDAS motif of &agr;Int1p. This peptide was synthesized by the Microchemical Facility of the University of Minnesota.
DHL DSF DRS YNH TEQ SI (SEQ ID NO:7)
F. A 17-mer peptide from the C-terminus of &agr;Int1p. This peptide was synthesized by the Microchemical Facility of the University of Minnesota.
WIQ NLQ EII YRN RFR RQ (SEQ ID NO:8)
Preparation and Evaluation of Antibodies
Polyclonal antibodies were prepared by Cocalico Biologics (Reamstown, Pa.) using the peptides B-F (SEQ ID NOS:4-8) listed above. Generally, each peptide is coupled to an adjuvant, the peptide-adjuvant mixture is injected into rabbits, and the rabbit receives booster injections of the same mixture every three-four weeks. Rabbit serum is withdrawn three weeks after the injections and tested for its titer against the original peptide.
One rabbit each was used to raise antibodies to each individual peptide. IgG antibodies were purified from the respective rabbit's antiserum by affinity purification on a Protein A-Sepharose column (BioRad) according to standard methods. In FIGS. 1 and 2, anti-Ca denotes antibodies raised to the 23-mer peptide (SEQ ID NO:5) encompassing the second divalent cation binding site; anti-RGD denotes antibodies to the 17-mer peptide encompassing the RGD site and flanking residues (SEQ ID NO:6). CAI-4 denotes the strain of C. albicans that was employed. Anti-Ca or anti-RGD antibodies in a concentration of 1.0 mg/ml were incubated with 1×106[35S]-methionine-labeled C. albicans blastospores for 30 minutes on ice at 4° C. Antibody-coated C. albicans blastospores were then incubated with confluent monolayers of HeLa cells in a 24-well microtiter plate for 60 minutes at 37° C. in 5% CO2, as described in a previous publication (Bendel and Hostetter, Journal of Clinical Investigation, 92, 1840-1849 (1993)). Removal of non-adherent C. alibicans blastospores, release of the HeLa monolayer with attached C. albicans blastospores, counting of the radiolabel, calculation of specific adhesion, and controls for non-specific adhesion were all performed according to the methods in the publication cited above. For FIG. 2, methods remained the same, save that CHO cell monolayers (Chinese hamster ovary cells, a second epithelial cell line) were substituted for HeLa cell monolayers.
FIG. 1 shows that the antibodies against the second divalent cation binding site (SEQ ID NO:5) or the RGD site and flanking residues (SEQ ID NO:6) inhibit binding to HeLa cells by about 50%. FIG. 2 shows that antibodies against the second divalent cation binding site or the RGD site inhibit binding to CHO cells by about 50%.
Induction of &agr;Int1p-Dependent Germ Tubes in Saccharomyces cerevisiae
The entire open reading frame of &agr;Int1 (BglII/SalI fragment) was subcloned into the plasmid pBM272 (obtained from Dr. James Bodley, University of Minnesota) after digestion with BamHI and SalI, in order to place the GAL1-10 promoter upstream of the &agr;Int1 start codon. This plasmid was named pCG01. S. cerevisiae YPH500, obtained from the Yeast Genetic Stock Center (Berkeley, Calif.), was transformed with pBM272 or pCG01 by the lithium acetate procedure as disclosed in Ito et al., J. Bacteriol., 153(1):163-168 (1983). Transformants were selected on agar-based minimal medium (MM=0.17% yeast nitrogen base/0.5% ammonium sulfate) with 2% glucose, in the absence of uracil. Induction of &agr;Int1 was achieved by growing transformants containing pCG01 to mid-exponential phase in non-inducing, non-repressing medium (MM without uracil with 2% raffinose) at 30° C., then harvesting, washing, and resuspending them in inducing medium (MM without uracil with 2% galactose) at 30° C. for the expression of &agr;Int1. YPH500 and YPH500 transformed with vector alone (pBM272) were grown under the identical conditions. S. cerevisiae transformants expressing &agr;Int1p from the plasmid pCG01 made abundant germ tubes after 6 hours' growth in inducing medium.
Applicants hereby incorporate-by-reference into the specification the accompanying Sequence Listing as required by 37 C.F.R. § 1.52(e)(5).
It will be appreciated by those skilled in the art that various modifications can be made to the above described embodiments of the invention without departing from the essential nature thereof. The invention is intended to encompass all such modifications within the scope of the appended claims. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference.12 5194 base pairs nucleic acid single linear DNA (genomic) 1 CCCAAAAAAG ATAAAATAAA AACAAAACAA AACAAAAGTA CTAACAAATT ATTGAAACTT 60 TTAATTTTTA ATAAAGAATC AGTAGATCTA TTGTTAAAAG AAATGAACTC AACTCCAAGT 120 AAATTATTAC CGATAGATAA ACATTCTCAT TTACAATTAC AGCCTCAATC GTCCTCGGCA 180 TCAATATTTA ATTCCCCAAC AAAACCATTG AATTTCCCCA GAACAAATTC CAAGCCGAGT 240 TTAGATCCAA ATTCAAGCTC TGATACCTAC ACTAGCGAAC AAGATCAAGA GAAAGGGAAA 300 GAAGAGAAAA AGGACACAGC CTTTCAAACA TCTTTTGATA GAAATTTTGA TCTTGATAAT 360 TCAATCGATA TACAACAAAC AATTCAACAT CAGCAACAAC AGCCACAACA ACAACAACAA 420 CTCTCACAAA CCGACAATAA TTTAATTGAT GAATTTTCTT TTCAAACACC GATGACTTCG 480 ACTTTAGACC TAACCAAGCA AAATCCAACT GTGGACAAAG TGAATGAAAA TCATGCACCA 540 ACTTATATAA ATACCTCCCC CAACAAATCA ATAATGAAAA AGGCAACTCC TAAAGCGTCA 600 CCTAAAAAAG TTGCATTTAC TGTAACTAAT CCCGAAATTC ATCATTATCC AGATAATAGA 660 GTCGAGGAAG AAGATCAAAG TCAACAAAAA GAAGATTCAG TTGAGCCACC CTTAATACAA 720 CATCAATGGA AAGATCCTTC TCAATTCAAT TATTCTGATG AAGATACAAA TGCTTCAGTT 780 CCACCAACAC CACCACTTCA TACGACGAAA CCTACTTTTG CGCAATTATT GAACAAAAAC 840 AACGAAGTCA ATCTGGAACC AGAGGCATTG ACAGATATGA AATTAAAGCG CGAAAATTTC 900 AGCAATTTAT CATTAGATGA AAAAGTCAAT TTATATCTTA GTCCCACTAA TAATAACAAT 960 AGTAAGAATG TGTCAGATAT GGATCTGCAT TTACAAAACT TGCAAGACGC TTCGAAAAAC 1020 AAAACTAATG AAAATATTCA CAATTTGTCA TTTGCTTTAA AAGCACCAAA GAATGATATT 1080 GAAAACCCAT TAAACTCATT GACTAACGCA GATATTCTGT TAAGATCATC TGGATCATCA 1140 CAATCGTCAT TACAATCTTT GAGGAATGAC AATCGTGTCT TGGAATCAGT GCCTGGGTCA 1200 CCTAAGAAGG TTAATCCTGG ATTGTCTTTG AATGACGGCA TAAAGGGGTT CTCTGATGAG 1260 GTTGTTGAAT CATTACTTCC TCGTGACTTA TCTCGAGACA AATTAGAGAC TACAAAAGAA 1320 CATGATGCAC CAGAACACAA CAATGAGAAT TTTATTGATG CTAAATCGAC TAATACCAAT 1380 AAGGGACAAC TCTTAGTATC ATCTGATGAT CATTTGGACT CTTTTGATAG ATCCTATAAC 1440 CACACTGAAC AATCAATTTT GAATCTTTTG AATAGTGCAT CACAATCTCA AATTTCGTTA 1500 AATGCATTGG AAAAACAAAG GCAAACACAG GAACAAGAAC AAACACAAGC GGCAGAGCCT 1560 GAAGAAGAAA CTTCGTTTAG TGATAATATC AAAGTTAAAC AAGAGCCAAA GAGCAATTTG 1620 GAGTTTGTCA AGGTTACCAT CAAGAAAGAA CCAGTTCTGG CCACGGAAAT AAAAGCTCCA 1680 AAAAGAGAAT TTTCAAGTCG AATATTAAGA ATAAAAAATG AAGATGAAAT TGCCGAACCA 1740 GCTGATATTC ATCCTAAAAA AGAAAATGAA GCAAACAGTC ATGTCGAAGA TACTGATGCA 1800 TTGTTGAAGA AAGCACTTAA TGATGATGAG GAATCTGACA CGACCCAAAA CTCAACGAAA 1860 ATGTCAATTC GTTTTCATAT TGATAGTGAT TGGAAATTGG AAGACAGTAA TGATGGCGAT 1920 AGAGAAGATA ATGATGATAT TTCTCGTTTT GAGAAATCAG ATATTTTGAA CGACGTATCA 1980 CAGACTTCTG ATATTATTGG TGACAAATAT GGAAACTCAT CAAGTGAAAT AACCACCAAA 2040 ACATTAGCAC CCCCAAGATC GGACAACAAT GACAAGGAGA ATTCTAAATC TTTGGAAGAT 2100 CCAGCTAATA ATGAATCATT GCAACAACAA TTGGAGGTAC CGCATACAAA AGAAGATGAT 2160 AGCATTTTAG CCAACTCGTC CAATATTGCT CCACCTGAAG AATTGACTTT GCCCGTAGTG 2220 GAAGCAAATG ATTATTCATC TTTTAATGAC GTGACCAAAA CTTTTGATGC ATACTCAAGC 2280 TTTGAAGAGT CATTATCTAG AGAGCACGAA ACTGATTCAA AACCAATTAA TTTCATATAA 2340 ATTTGGCATA AACAAGAAAA GCAGAAGAAA CATCAAATTC ATAAAGTTCC AACTAAACAG 2400 ATCATTGCTA GTTATCAACA ATACAAAAAC GAACAAGAAT CTCGTGTTAC TAGTGATAAA 2460 GTGAAAATCC CAAATGCCAT ACAATTCAAG AAATTCAAAG AGGTAAATGT CATGTCAAGA 2520 AGAGTTGTTA GTCCAGACAT GGATGATTTG AATGTATCTC AATTTTTACC AGAATTATCT 2580 GAAGACTCTG GATTTAAAGA TTTGAATTTT GCCAACTACT CCAATAACAC CAACAGACCA 2640 AGAAGTTTTA CTCCATTGAG CACTAAAAAT GTCTTGTCGA ATATTGATAA CGATCCTAAT 2700 GTTGTTGAAC CTCCTGAACC GAAATCATAT GCTGAAATTA GAAATGCTAG ACGGTTATCA 2760 GCTAATAAGG CAGCGCCAAA TCAGGCACCA CCATTGCCAC CACAACGACA ACCATCTTCA 2820 ACTCGTTCCA ATTCAAATAA ACGAGTGTCC AGATTTAGAG TGCCCACATT TGAAATTAGA 2880 AGAACTTCTT CAGCATTAGC ACCTTGTGAC ATGTATAATG ATATTTTTGA TGATTTCGGT 2940 GCGGGTTCTA AACCAACTAT AAAGGCAGAA GGAATGAAAA CATTGCCAAG TATGGATAAA 3000 GATGATGTCA AGAGGATTTT GAATGCAAAG AAAGGTGTGA CTCAAGATGA ATATATAAAT 3060 GCCAAACTTG TTGATCAAAA ACCTAAAAAG AATTCAATTG TCACCGATCC CGAAGACCGA 3120 TATGAAGAAT TACAACAAAC TGCCTCTATA CACAATGCCA CCATTGATTC AAGTATTTAT 3180 GGCCGACCAG ACTCCATTTC TACCGACATG TTGCCTTATC TTAGTGATGA ATTGAAAAAA 3240 CCACCTACGG CTTTATTATC TGCTGATCGT TTGTTTATGG AACAAGAAGT ACATCCGTTA 3300 AGATCAAACT CTGTTTTGGT TCACCCAGGG GCAGGAGCAG CAACTAATTC TTCAATGTTA 3360 CCAGAGCCAG ATTTTGAATT AATCAATTCA CCTGCTAGAA ATGTGCTGAA CAACAGTGAT 3420 AATGTCGCCA TCAGTGGTAA TGCTAGTACT ATTAGTTTTA ACCAATTGGA TATGAATTTT 3480 GATGACCAAG CTACAATTGG TCAAAAAATC CAAGAGCAAC CTGCTTCAAA ATCCGCCAAT 3540 ACTGTTCGTG GTGATGATGA TGGATTGGCC AGTGCACCTG AAACACCAAG AACTCCTACC 3600 AAAAAGGAGT CCATATCAAG CAAGCCTGCC AAGCTTTCTT CTGCCTCCCC TAGAAAATCA 3660 CCAATTAAGA TTGGTTCACC AGTTCGAGTT ATTAAGAAAA ATGGATCAAT TGCTGGCATT 3720 GAACCAATCC CAAAAGCCAC TCACAAACCG AAGAAATCAT TCCAAGGAAA CGAGATTTCA 3780 AACCATAAAG TACGAGATGG TGGAATTTCA CCAAGCTCCG GATCAGAGCA TCAACAGCAT 3840 AATCCTAGTA TGGTTTCTGT TCCTTCACAG TATACTGATG CTACTTCAAC GGTTCCAGAT 3900 GAAAACAAAG ATGTTCAACA CAAGCCTCGT GAAAAGCAAA AGCAAAAGCA TCACCATCGC 3960 CATCATCATC ATCATCATAA ACAAAAAACT GATATTCCGG GTGTTGTTGA TGATGAAATT 4020 CCTGATGTAG GATTACAAGA ACGAGGCAAA TTATTCTTTA GAGTTTTAGG AATTAAGAAT 4080 ATCAATTTAC CCGATATTAA TACTCACAAA GGAAGATTCA CTTTAACGTT GGATAATGGA 4140 GTGCATTGTG TTACTACACC AGAATACAAC ATGGACGACC ATAATGTTGC CATAGGTAAA 4200 GAATTTGAGT TGACAGTTGC TGATTCATTA GAGTTTATTT TAACTTTGAA GGCATCATAT 4260 GAAAAACCTC GTGGTACATT AGTAGAAGTG ACTGAAAAGA AAGTTGTCAA ATCAAGAAAT 4320 AGATTGAGTC GATTATTTGG ATCGAAAGAT ATTATCACCA CGACAAAGTT TGTGCCCACT 4380 GAAGTCAAAG ATACCTGGGC TAATAAGTTT GCTCCTGATG GTTCATTTGC TAGATGTTAC 4440 ATTGATTTAC AACAATTTGA AGACCAAATC ACCGGTAAAG CATCACAGTT TGATCTCAAT 4500 TGTTTTAATG AATGGGAAAC TATGAGTAAT GGCAATCAAC CAATGAAAAG AGGCAAACCT 4560 TATAAGATTG CTCAATTGGA AGTTAAAATG TTGTATGTTC CACGATCAGA TCCAAGAGAA 4620 ATATTACCAA CCAGCATTAG ATCCGCATAT GAAAGCATCA ATGAATTAAA CAATGAACAG 4680 AATAATTACT TTGAAGGTTA TTTACATCAA GAAGGAGGTG ATTGTCCAAT TTTTAAGAAA 4740 CGTTTTTTCA AATTAATGGG CACTTCTTTA TTGGCTCATA GTGAAATATC TCATAAAACT 4800 AGAGCCAAAA TTAATTTATC AAAAGTTGTT GATTTGATTT ATGTTGATAA AGAAAACATT 4860 GATCGTTCCA ATCATCGAAA TTTCAGTGAT GTGTTATTGT TGGATCATGC ATTCAAAATC 4920 AAATTTGCTA ATGGTGAGTT GATTGATTTT TGTGCTCCTA ATAAACATGA AATGAAAATA 4980 TGGATTCAAA ATTTACAAGA AATTATCTAT AGAAATCGGT TCAGACGTCA ACCATGGGTA 5040 AATTTGATGC TTCAACAACA ACAACAACAA CAACAACAAC AAAGCTCCCA ACAGTAATTG 5100 AAAGGTCTAC TTTTGATTTT TTTAATTTTA ATTGGCAAAT ATATGCCCAT TTTGTATTAT 5160 CTTTTAGTCT AATAGCGTTT TCTTTTTTTC CAGT 5194 1664 amino acids amino acid single linear protein 2 Met Asn Ser Thr Pro Ser Lys Leu Leu Pro Ile Asp Lys His Ser His 1 5 10 15 Leu Gln Leu Gln Pro Gln Ser Ser Ser Ala Ser Ile Phe Asn Ser Pro 20 25 30 Thr Lys Pro Leu Asn Phe Pro Arg Thr Asn Ser Lys Pro Ser Leu Asp 35 40 45 Pro Asn Ser Ser Ser Asp Thr Tyr Thr Ser Glu Gln Asp Gln Glu Lys 50 55 60 Gly Lys Glu Glu Lys Lys Asp Thr Ala Phe Gln Thr Ser Phe Asp Arg 65 70 75 80 Asn Phe Asp Leu Asp Asn Ser Ile Asp Ile Gln Gln Thr Ile Gln His 85 90 95 Gln Gln Gln Gln Pro Gln Gln Gln Gln Gln Leu Ser Gln Thr Asp Asn 100 105 110 Asn Leu Ile Asp Glu Phe Ser Phe Gln Thr Pro Met Thr Ser Thr Leu 115 120 125 Asp Leu Thr Lys Gln Asn Pro Thr Val Asp Lys Val Asn Glu Asn His 130 135 140 Ala Pro Thr Tyr Ile Asn Thr Ser Pro Asn Lys Ser Ile Met Lys Lys 145 150 155 160 Ala Thr Pro Lys Ala Ser Pro Lys Lys Val Ala Phe Thr Val Thr Asn 165 170 175 Pro Glu Ile His His Tyr Pro Asp Asn Arg Val Glu Glu Glu Asp Gln 180 185 190 Ser Gln Gln Lys Glu Asp Ser Val Glu Pro Pro Leu Ile Gln His Gln 195 200 205 Trp Lys Asp Pro Ser Gln Phe Asn Tyr Ser Asp Glu Asp Thr Asn Ala 210 215 220 Ser Val Pro Pro Thr Pro Pro Leu His Thr Thr Lys Pro Thr Phe Ala 225 230 235 240 Gln Leu Leu Asn Lys Asn Asn Glu Val Asn Ser Glu Pro Glu Ala Leu 245 250 255 Thr Asp Met Lys Leu Lys Arg Glu Asn Phe Ser Asn Leu Ser Leu Asp 260 265 270 Glu Lys Val Asn Leu Tyr Leu Ser Pro Thr Asn Asn Asn Asn Ser Lys 275 280 285 Asn Val Ser Asp Met Asp Ser His Leu Gln Asn Leu Gln Asp Ala Ser 290 295 300 Lys Asn Lys Thr Asn Glu Asn Ile His Asn Leu Ser Phe Ala Leu Lys 305 310 315 320 Ala Pro Lys Asn Asp Ile Glu Asn Pro Leu Asn Ser Leu Thr Asn Ala 325 330 335 Asp Ile Ser Leu Arg Ser Ser Gly Ser Ser Gln Ser Ser Leu Gln Ser 340 345 350 Leu Arg Asn Asp Asn Arg Val Leu Glu Ser Val Pro Gly Ser Pro Lys 355 360 365 Lys Val Asn Pro Gly Leu Ser Leu Asn Asp Gly Ile Lys Gly Phe Ser 370 375 380 Asp Glu Val Val Glu Ser Leu Leu Pro Arg Asp Leu Ser Arg Asp Lys 385 390 395 400 Leu Glu Thr Thr Lys Glu His Asp Ala Pro Glu His Asn Asn Glu Asn 405 410 415 Phe Ile Asp Ala Lys Ser Thr Asn Thr Asn Lys Gly Gln Leu Leu Val 420 425 430 Ser Ser Asp Asp His Leu Asp Ser Phe Asp Arg Ser Tyr Asn His Thr 435 440 445 Glu Gln Ser Ile Leu Asn Leu Leu Asn Ser Ala Ser Gln Ser Gln Ile 450 455 460 Ser Leu Asn Ala Leu Glu Lys Gln Arg Gln Thr Gln Glu Gln Glu Gln 465 470 475 480 Thr Gln Ala Ala Glu Pro Glu Glu Glu Thr Ser Phe Ser Asp Asn Ile 485 490 495 Lys Val Lys Gln Glu Pro Lys Ser Asn Leu Glu Phe Val Lys Val Thr 500 505 510 Ile Lys Lys Glu Pro Val Ser Ala Thr Glu Ile Lys Ala Pro Lys Arg 515 520 525 Glu Phe Ser Ser Arg Ile Leu Arg Ile Lys Asn Glu Asp Glu Ile Ala 530 535 540 Glu Pro Ala Asp Ile His Pro Lys Lys Glu Asn Glu Ala Asn Ser His 545 550 555 560 Val Glu Asp Thr Asp Ala Leu Leu Lys Lys Ala Leu Asn Asp Asp Glu 565 570 575 Glu Ser Asp Thr Thr Gln Asn Ser Thr Lys Met Ser Ile Arg Phe His 580 585 590 Ile Asp Ser Asp Trp Lys Leu Glu Asp Ser Asn Asp Gly Asp Arg Glu 595 600 605 Asp Asn Asp Asp Ile Ser Arg Phe Glu Lys Ser Asp Ile Leu Asn Asp 610 615 620 Val Ser Gln Thr Ser Asp Ile Ile Gly Asp Lys Tyr Gly Asn Ser Ser 625 630 635 640 Ser Glu Ile Thr Thr Lys Thr Leu Ala Pro Pro Arg Ser Asp Asn Asn 645 650 655 Asp Lys Glu Asn Ser Lys Ser Leu Glu Asp Pro Ala Asn Asn Glu Ser 660 665 670 Leu Gln Gln Gln Leu Glu Val Pro His Thr Lys Glu Asp Asp Ser Ile 675 680 685 Leu Ala Asn Ser Ser Asn Ile Ala Pro Pro Glu Glu Leu Thr Leu Pro 690 695 700 Val Val Glu Ala Asn Asp Tyr Ser Ser Phe Asn Asp Val Thr Lys Thr 705 710 715 720 Phe Asp Ala Tyr Ser Ser Phe Glu Glu Ser Leu Ser Arg Glu His Glu 725 730 735 Thr Asp Ser Lys Pro Ile Asn Phe Ile Ser Ile Trp His Lys Gln Glu 740 745 750 Lys Gln Lys Lys His Gln Ile His Lys Val Pro Thr Lys Gln Ile Ile 755 760 765 Ala Ser Tyr Gln Gln Tyr Lys Asn Glu Gln Glu Ser Arg Val Thr Ser 770 775 780 Asp Lys Val Lys Ile Pro Asn Ala Ile Gln Phe Lys Lys Phe Lys Glu 785 790 795 800 Val Asn Val Met Ser Arg Arg Val Val Ser Pro Asp Met Asp Asp Leu 805 810 815 Asn Val Ser Gln Phe Leu Pro Glu Leu Ser Glu Asp Ser Gly Phe Lys 820 825 830 Asp Leu Asn Phe Ala Asn Tyr Ser Asn Asn Thr Asn Arg Pro Arg Ser 835 840 845 Phe Thr Pro Leu Ser Thr Lys Asn Val Leu Ser Asn Ile Asp Asn Asp 850 855 860 Pro Asn Val Val Glu Pro Pro Glu Pro Lys Ser Tyr Ala Glu Ile Arg 865 870 875 880 Asn Ala Arg Arg Leu Ser Ala Asn Lys Ala Ala Pro Asn Gln Ala Pro 885 890 895 Pro Leu Pro Pro Gln Arg Gln Pro Ser Ser Thr Arg Ser Asn Ser Asn 900 905 910 Lys Arg Val Ser Arg Phe Arg Val Pro Thr Phe Glu Ile Arg Arg Thr 915 920 925 Ser Ser Ala Leu Ala Pro Cys Asp Met Tyr Asn Asp Ile Phe Asp Asp 930 935 940 Phe Gly Ala Gly Ser Lys Pro Thr Ile Lys Ala Glu Gly Met Lys Thr 945 950 955 960 Leu Pro Ser Met Asp Lys Asp Asp Val Lys Arg Ile Leu Asn Ala Lys 965 970 975 Lys Gly Val Thr Gln Asp Glu Tyr Ile Asn Ala Lys Leu Val Asp Gln 980 985 990 Lys Pro Lys Lys Asn Ser Ile Val Thr Asp Pro Glu Asp Arg Tyr Glu 995 1000 1005 Glu Leu Gln Gln Thr Ala Ser Ile His Asn Ala Thr Ile Asp Ser Ser 1010 1015 1020 Ile Tyr Gly Arg Pro Asp Ser Ile Ser Thr Asp Met Leu Pro Tyr Leu 1025 1030 1035 1040 Ser Asp Glu Leu Lys Lys Pro Pro Thr Ala Leu Leu Ser Ala Asp Arg 1045 1050 1055 Leu Phe Met Glu Gln Glu Val His Pro Leu Arg Ser Asn Ser Val Leu 1060 1065 1070 Val His Pro Gly Ala Gly Ala Ala Thr Asn Ser Ser Met Leu Pro Glu 1075 1080 1085 Pro Asp Phe Glu Leu Ile Asn Ser Pro Ala Arg Asn Val Ser Asn Asn 1090 1095 1100 Ser Asp Asn Val Ala Ile Ser Gly Asn Ala Ser Thr Ile Ser Phe Asn 1105 1110 1115 1120 Gln Leu Asp Met Asn Phe Asp Asp Gln Ala Thr Ile Gly Gln Lys Ile 1125 1130 1135 Gln Glu Gln Pro Ala Ser Lys Ser Ala Asn Thr Val Arg Gly Asp Asp 1140 1145 1150 Asp Gly Leu Ala Ser Ala Pro Glu Thr Pro Arg Thr Pro Thr Lys Lys 1155 1160 1165 Glu Ser Ile Ser Ser Lys Pro Ala Lys Leu Ser Ser Ala Ser Pro Arg 1170 1175 1180 Lys Ser Pro Ile Lys Ile Gly Ser Pro Val Arg Val Ile Lys Lys Asn 1185 1190 1195 1200 Gly Ser Ile Ala Gly Ile Glu Pro Ile Pro Lys Ala Thr His Lys Pro 1205 1210 1215 Lys Lys Ser Phe Gln Gly Asn Glu Ile Ser Asn His Lys Val Arg Asp 1220 1225 1230 Gly Gly Ile Ser Pro Ser Ser Gly Ser Glu His Gln Gln His Asn Pro 1235 1240 1245 Ser Met Val Ser Val Pro Ser Gln Tyr Thr Asp Ala Thr Ser Thr Val 1250 1255 1260 Pro Asp Glu Asn Lys Asp Val Gln His Lys Pro Arg Glu Lys Gln Lys 1265 1270 1275 1280 Gln Lys His His His Arg His His His His His His Lys Gln Lys Thr 1285 1290 1295 Asp Ile Pro Gly Val Val Asp Asp Glu Ile Pro Asp Val Gly Leu Gln 1300 1305 1310 Glu Arg Gly Lys Leu Phe Phe Arg Val Leu Gly Ile Lys Asn Ile Asn 1315 1320 1325 Leu Pro Asp Ile Asn Thr His Lys Gly Arg Phe Thr Leu Thr Leu Asp 1330 1335 1340 Asn Gly Val His Cys Val Thr Thr Pro Glu Tyr Asn Met Asp Asp His 1345 1350 1355 1360 Asn Val Ala Ile Gly Lys Glu Phe Glu Leu Thr Val Ala Asp Ser Leu 1365 1370 1375 Glu Phe Ile Leu Thr Leu Lys Ala Ser Tyr Glu Lys Pro Arg Gly Thr 1380 1385 1390 Leu Val Glu Val Thr Glu Lys Lys Val Val Lys Ser Arg Asn Arg Leu 1395 1400 1405 Ser Arg Leu Phe Gly Ser Lys Asp Ile Ile Thr Thr Thr Lys Phe Val 1410 1415 1420 Pro Thr Glu Val Lys Asp Thr Trp Ala Asn Lys Phe Ala Pro Asp Gly 1425 1430 1435 1440 Ser Phe Ala Arg Cys Tyr Ile Asp Leu Gln Gln Phe Glu Asp Gln Ile 1445 1450 1455 Thr Gly Lys Ala Ser Gln Phe Asp Leu Asn Cys Phe Asn Glu Trp Glu 1460 1465 1470 Thr Met Ser Asn Gly Asn Gln Pro Met Lys Arg Gly Lys Pro Tyr Lys 1475 1480 1485 Ile Ala Gln Leu Glu Val Lys Met Leu Tyr Val Pro Arg Ser Asp Pro 1490 1495 1500 Arg Glu Ile Leu Pro Thr Ser Ile Arg Ser Ala Tyr Glu Ser Ile Asn 1505 1510 1515 1520 Glu Leu Asn Asn Glu Gln Asn Asn Tyr Phe Glu Gly Tyr Leu His Gln 1525 1530 1535 Glu Gly Gly Asp Cys Pro Ile Phe Lys Lys Arg Phe Phe Lys Leu Met 1540 1545 1550 Gly Thr Ser Leu Leu Ala His Ser Glu Ile Ser His Lys Thr Arg Ala 1555 1560 1565 Lys Ile Asn Leu Ser Lys Val Val Asp Leu Ile Tyr Val Asp Lys Glu 1570 1575 1580 Asn Ile Asp Arg Ser Asn His Arg Asn Phe Ser Asp Val Leu Leu Leu 1585 1590 1595 1600 Asp His Ala Phe Lys Ile Lys Phe Ala Asn Gly Glu Leu Ile Asp Phe 1605 1610 1615 Cys Ala Pro Asn Lys His Glu Met Lys Ile Trp Ile Gln Asn Leu Gln 1620 1625 1630 Glu Ile Ile Tyr Arg Asn Arg Phe Arg Arg Gln Pro Trp Val Asn Leu 1635 1640 1645 Met Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ser Ser Gln Gln 1650 1655 1660 236 amino acids amino acid single linear protein amino acid positions 218-453 from SEQ ID NO2 3 Ser Asp Glu Asp Thr Asn Ala Ser Val Pro Pro Thr Pro Pro Leu His 1 5 10 15 Thr Thr Lys Pro Thr Phe Ala Gln Leu Leu Asn Lys Asn Asn Glu Val 20 25 30 Asn Ser Glu Pro Glu Ala Leu Thr Asp Met Lys Leu Lys Arg Glu Asn 35 40 45 Phe Ser Asn Leu Ser Leu Asp Glu Lys Val Asn Leu Tyr Leu Ser Pro 50 55 60 Thr Asn Asn Asn Asn Ser Lys Asn Val Ser Asp Met Asp Ser His Leu 65 70 75 80 Gln Asn Leu Gln Asp Ala Ser Lys Asn Lys Thr Asn Glu Asn Ile His 85 90 95 Asn Leu Ser Phe Ala Leu Lys Ala Pro Lys Asn Asp Ile Glu Asn Pro 100 105 110 Leu Asn Ser Leu Thr Asn Ala Asp Ile Ser Leu Arg Ser Ser Gly Ser 115 120 125 Ser Gln Ser Ser Leu Gln Ser Leu Arg Asn Asp Asn Arg Val Leu Glu 130 135 140 Ser Val Pro Gly Ser Pro Lys Lys Val Asn Pro Gly Leu Ser Leu Asn 145 150 155 160 Asp Gly Ile Lys Gly Phe Ser Asp Glu Val Val Glu Ser Leu Leu Pro 165 170 175 Arg Asp Leu Ser Arg Asp Lys Leu Glu Thr Thr Lys Glu His Asp Ala 180 185 190 Pro Glu His Asn Asn Glu Asn Phe Ile Asp Ala Lys Ser Thr Asn Thr 195 200 205 Asn Lys Gly Gln Leu Leu Val Ser Ser Asp Asp His Leu Asp Ser Phe 210 215 220 Asp Arg Ser Tyr Asn His Thr Glu Gln Ser Ile Leu 225 230 235 23 amino acids amino acid single linear peptide 4 Tyr Leu Ser Pro Thr Asn Asn Asn Asn Ser Lys Asn Val Ser Asp Met 1 5 10 15 Asp Leu His Leu Gln Asn Leu 20 23 amino acids amino acid single linear peptide 5 Asp Trp Lys Leu Glu Asp Ser Asn Asp Gly Asp Arg Glu Asp Asn Asp 1 5 10 15 Asp Ile Ser Arg Phe Glu Lys 20 17 amino acids amino acid single linear peptide 6 Ser Lys Ser Ala Asn Thr Val Arg Gly Asp Asp Asp Gly Leu Ala Ser 1 5 10 15 Ala 17 amino acids amino acid single linear peptide 7 Asp His Leu Asp Ser Phe Asp Arg Ser Tyr Asn His Thr Glu Gln Ser 1 5 10 15 Ile 17 amino acids amino acid single linear peptide 8 Trp Ile Gln Asn Leu Gln Glu Ile Ile Tyr Arg Asn Arg Phe Arg Arg 1 5 10 15 Gln 20 base pairs nucleic acid single linear DNA (genomic) 9 GAATTCAATG CTACCCTCAA 20 20 base pairs nucleic acid single linear DNA (genomic) 10 CCCGGGGGAC CCCCTTCACT 20 18 base pairs nucleic acid single linear DNA (genomic) 11 AARGTYGGWT TYTTYAAR 18 18 base pairs nucleic acid single linear DNA (genomic) 12 GAAATHGAYG AYTTRATG 18
1. An isolated and purified antibody to a Candida albicans integrin-like protein, wherein the protein has an amino acid sequence having SEQ ID NO:2, and wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
2. The antibody of claim 1 wherein the antibody is monoclonal or polyclonal.
3. The antibody of claim 1 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
4. The antibody of claim 1 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
5. The antibody of claim 1 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
6. An isolated and purified antibody to a polypeptide, wherein the polypeptide has the amino acid sequence of SEQ ID NO:3, and wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
7. The antibody of claim 6 wherein the antibody is monoclonal or polyclonal.
8. The antibody of claim 6, wherein the antibody blocks adhesion to epithelial and/or endotheial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
9. The antibody of claim 6 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
10. The antibody of claim 6 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
11. An isolated and purified antibody to a polypeptide, wherein the polypeptide consists of SEQ ID NO:3, and wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
12. The antibody of claim 11 wherein the antibody is monoclonal or polyclonal.
13. The antibody of claim 11 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
14. The antibody of claim 11 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
15. The antibody of claim 11 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
16. An isolated and purified antibody to a peptide consisting of an amino acid sequence selected from the group consisting of:
- (a) YLS PTN NNN SKN VSD MDL HLQ NL (SEQ ID NO:4);
- (b) DWK LED SND GDR EDN DDI SRF EK (SEQ ID NO:5);
- (c) SKS ANT VRG DDD GLA SA (SEQ ID NO:6);
- (d) DHL DSF DRS YNH TEQ SI (SEQ ID NO:7); and
- (e) WIQ NLQ EII YRN RFR RQ (SEQ ID NO:8).
17. The antibody of claim 16 wherein the antibody is monoclonal or polyclonal.
18. The antibody of claim 16 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
19. The antibody of claim 16 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
20. The antibody of claim 16 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
21. The antibody of claim 16 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
22. An isolated and purified antibody to a peptide having of an amino acid sequence selected from the group consisting of:
- (a) YLS PTN NNN SKN VSD MDL HLQ NL (SEQ ID NO:4);
- (b) DWK LED SND GDR EDN DDI SRF EK (SEQ ID NO:5);
- (c) SKS ANT VRG DDD GLA SA (SEQ ID NO:6);
- (d) DHL DSF DRS YNH TEQ SI (SEQ II) NO:7); and
- (e) WIQ NLQ EII YRN RFR RQ (SEQ ID NO:8), wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
23. The antibody of claim 22 wherein the antibody is monoclonal or polyclonal.
24. The antibody of claim 22 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
25. The antibody of claim 22 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
26. The antibody of claim 22 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
27. An isolated and purified antibody to a polypeptide with integrin-like motifs encoded by a polynucleotide that hybridizes to DNA complementary to DNA having SEQ ID NO:1 under stringency conditions of hybridization in buffer containing 5×SSC, 5× Denhardt's, 0.5% SDS, 1 mg salmon sperm/25 mls of hybridization solution incubated at 65° C. overnight, followed by high stringency washing with 0.2×SSC/0.1% SDS at 65° C., wherein the polypeptide with integrin-like motifs contains an I domain, two EF-hand divalent cation binding sites, a sequence sufficient to form a transmembrane domain, an internal RGD tripeptide, and a carboxy-terminal sequence having a single tyrosine residue, and wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
28. The antibody of claim 27 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
29. The antibody of claim 27 wherein the antibody is monoclonal or polyclonal.
30. The antibody of claim 27 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
31. The antibody of claim 27 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
32. The antibody of claim 27 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
33. An isolated and purified antibody to a Candida albicans polypeptide encoded by a polynucleotide having SEQ ID NO:1, wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells.
34. The antibody of claim 33 wherein the antibody is monoclonal or polyclonal.
35. The antibody of claim 33 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 30 percent.
36. The antibody of claim 33 wherein the antibody blocks Candida albicans adhesion to epithelial and/or endothelial cells by at least about 50 percent.
37. The antibody of claim 33 wherein the antibody blocks adhesion to epithelial and/or endothelial cells by Candida albicans selected from a morphological stage of Candida albicans development selected from the group consisting of blastospores, germ tubes, and hyphae.
|4542020||September 17, 1985||Jackson et al.|
|4661454||April 28, 1987||Botstein et al.|
|4670382||June 2, 1987||Buckley et al.|
|4735901||April 5, 1988||Kurtz et al.|
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|5886151||March 23, 1999||Hostetter et al.|
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|278 840||July 1994||CZ|
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Filed: Oct 15, 2001
Date of Patent: Aug 10, 2004
Patent Publication Number: 20030082680
Assignee: Regents of the University of Minnesota (Minneapolis, MN)
Inventors: Margaret K. Hostetter (Milford, CT), Cheryl A. Gale (Maple Grove, MN), Kathleen Kendrick (Columbus, OH)
Primary Examiner: Elizabeth Kemmerer
Assistant Examiner: Claire M. Kaufman
Attorney, Agent or Law Firm: Mueting, Raasch & Gebhardt, P.A.
Application Number: 09/978,343
International Classification: C07K/1614; C07K/1628;